Germplasm cryobanking is an effective way of preserving scientifically, medically, economically and environmentally important genetic stocks indefinitely. Although rats are important system for modeling human disease, there is no established germplasm cryopreservation protocol which would allow cost-effective management of rat genetic resources. Collection of epididymal sperm is the simplest and most cost-effective method of archiving genetics for future use. However, to date, there is only one protocol showing only 10% post-thaw motility. Thus, there is an urgent need for understanding the physiological and cryobiological factors affecting rat sperm in order to improve the rat sperm cryosurvival. The goal of this application is to develop freezing protocols for epididymal rat sperm which would allow reconstitution of genetics by using standard artificial insemination and in-vitro fertilization methods. Over the last few years, our laboratory has generated ample amount of data related with optimal sperm handling, Percoll separation, freezing extender and cooling and warming conditions for the rat sperm. These would help reduce the injuries caused by physical, hypothermic, osmotic stress and lethal ice formation, and ultimately yield acceptable post-thaw recovery rate. The data which have been collected in our laboratory in combination with proposed cryobiologic studies outlined in this application will allow us to develop efficient and reliable cryopreservation protocol for epididymal rat sperm. PUBLIC HEALTH RELEVANCE (provided by applicant): This proposal aims at developing efficient cryopreservation method for rat sperm which would be used to reconstitute genetically important rat strains through commonly used assisted reproductive technologies such as artificial insemination or in-vitro fertilization if needed. The proposed studies will therefore address to the biomedical community by providing a reliable protocol for rat sperm cryopreservation in order to avoid continuous live animal housing which is a significant burden for National Institutes of Health and its investigators around the nation.